A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia

Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany.
PLoS Biology (Impact Factor: 11.77). 06/2010; 8(6):e1000408. DOI: 10.1371/journal.pbio.1000408
Source: PubMed

ABSTRACT DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR) have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA) screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP). We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair.

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Available from: Mirko Theis, Aug 21, 2015
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    • "Disruption of spg11 (SPG11, spatacsin) or zfyve26 (SPG15, spastizin) expression during zebrafish development induced a range of developmental defects, including locomotor impairment and abnormal architecture of spinal MN axons (Martin et al., 2012; Southgate et al., 2010). These two proteins are components of a multi-protein complex (Słabicki et al., 2010) and AMOs targeting the two genes at the same time suggested that they were involved in a pathway required for spinal MN axon outgrowth (Martin et al., 2012). However, an EMS (Raldú a et al., 2008) was apparently induced by changing the expression of these genes, thus making it difficult to interpret the phenotype, which may also be due to a developmental delay. "
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    ABSTRACT: Motor neuron diseases (MNDs) are an etiologically heterogeneous group of disorders of neurodegenerative origin, which result in degeneration of lower (LMNs) and/or upper motor neurons (UMNs). Neurodegenerative MNDs include pure hereditary spastic paraplegia (HSP), which involves specific degeneration of UMNs, leading to progressive spasticity of the lower limbs. In contrast, spinal muscular atrophy (SMA) involves the specific degeneration of LMNs, with symmetrical muscle weakness and atrophy. Amyotrophic lateral sclerosis (ALS), the most common adult-onset MND, is characterized by the degeneration of both UMNs and LMNs, leading to progressive muscle weakness, atrophy, and spasticity. A review of the comparative neuroanatomy of the human and zebrafish motor systems showed that, while the zebrafish was a homologous model for LMN disorders, such as SMA, it was only partially relevant in the case of UMN disorders, due to the absence of corticospinal and rubrospinal tracts in its central nervous system. Even considering the limitation of this model to fully reproduce the human UMN disorders, zebrafish offer an excellent alternative vertebrate model for the molecular and genetic dissection of MND mechanisms. Its advantages include the conservation of genome and physiological processes and applicable in vivo tools, including easy imaging, loss or gain of function methods, behavioral tests to examine changes in motor activity, and the ease of simultaneous chemical/drug testing on large numbers of animals. This facilitates the assessment of the environmental origin of MNDs, alone or in combination with genetic traits and putative modifier genes. Positive hits obtained by phenotype-based small-molecule screening using zebrafish may potentially be effective drugs for treatment of human MNDs.
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    • "Consequently, the competition between homologous recombination and NHEJ must be considered when developing therapeutic applications based on the generation of specific DSBs using molecular scissors. Using RNAi, it was recently shown that the absence of proteins involved in non-conservative NHEJ DSB repair augments the frequency of repair events mediated by the conservative homologous recombination pathway in mammalian cells (Slabicki et al., 2010). This observation suggests that inhibition of NHEJ may favor DSB repair via the conservative mechanism. "
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    ABSTRACT: The engineering of protein-DNA interactions in different protein scaffolds may provide "toolkits" to modify the genome. Homing endonucleases are powerful tools for genome manipulation through homologous recombination, as these enzymes possess a very low frequency of DNA cleavage in eukaryotic genomes due to their high specificity. Therefore, the combination of a precise "cutter" with the presence of a natural or modified homologous DNA donor provides a potentially useful means to modify the genome. However, the basis of protein-DNA recognition must be understood to generate tailored enzymes that target the DNA at sites of interest. The engineering of homing endonucleases and alternative scaffolds, such as zinc fingers or transcription activator-like effector domains, has demonstrated the potential of these approaches to create new specific instruments to target genes for inactivation or repair. Customized homing endonucleases targeting selected human genes can excise or correct regions of genes implicated in monogenic diseases, thereby representing important tools for intervention in eukaryotic genomes.
    Critical Reviews in Biochemistry and Molecular Biology 01/2012; 47(3):207-21. DOI:10.3109/10409238.2011.652358 · 5.81 Impact Factor
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    • "Several genomewide RNAi screening analyses have recently improved our understanding of the DNA repair pathway [52] [53] [54]. Słabicki et al. identified 61 genes affecting DNA DSB repair in human HeLa cells [54]. The downregulation of 17 of these genes led to an increase in endonuclease-induced homologous recombination. "
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    ABSTRACT: Homologous gene targeting (HGT) is a precise but inefficient process for genome engineering. Several methods for increasing its efficiency have been developed, including the use of rare cutting endonucleases. However, there is still room for improvement, as even nuclease-induced HGT may vary in efficiency as a function of the nuclease, target site, and cell type considered. We have developed a high-throughput screening assay for the identification of factors stimulating meganuclease-induced HGT. We used this assay to explore a collection of siRNAs targeting 19,121 human genes. At the end of secondary screening, we had identified 64 genes for which knockdown affected nuclease-induced HGT. Two of the strongest candidates were characterized further. We showed that siRNAs directed against the ATF7IP gene, encoding a protein involved in chromatin remodeling, stimulated HGT by a factor of three to eight, at various loci and in different cell types. This method thus led to the identification of a number of genes, the manipulation of which might increase rates of targeted recombination.
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